The continuous advances in molecular biology, biotechnology and clinical research have resulted in an ever increasing number of uses for DNA, RNA and proteins. For example, polymerase chain reaction (PCR) technology has dramatically expanded the use of DNA and RNA in basic research, in clinical diagnostics such as detection of messenger RNA (mRNA) by reverse transcription PCR (RT-PCR), and the use of PCR in detection of genetic defects. In the protein field, identification of proteins by Western blotting has become an important tool in studying gene expression in basic research and identification of specific proteins for diagnostic purposes, as exemplified by viral protein detection.
The increased use of RNA, DNA and proteins has created a need for fast, simple and reliable methods and reagents for isolating DNA, RNA and proteins. In many applications, collecting the biological material sample and subsequent analysis thereof would be substantially simplified if the three cellular components (RNA, DNA and proteins) could be simultaneously isolated from a single sample. The simultaneous isolation is especially important when the sample size is so small, such as in biopsy, that it precludes its separation into smaller samples to perform separate isolation protocols for DNA, RNA and proteins.
There are known methods for isolating DNA, RNA and proteins from a single biological material sample. One such method is described in Coombs, L. M., et al.: "Simultaneous Isolation of DNA, RNA and Antigenic Protein Exhibiting Kinase Activity from Small Tumor Samples Using Guanidine Isothiocyanate", Anal. Biochem., 188, 338-343 (1990). The Coombs et al. method is based on ultracentrifugation of the sample homogenate in a guanidine-cesium chloride solution. The sample is homogenized in 4M guanidine thiocyanate and then overlayered on a cesium chloride (CsCl) solution and centrifuged at&gt;100,000 g for about 18 hours. Following centrifugation, DNA, RNA and proteins are separated and purified over the next 12-24 hours. This method has several limitations or drawbacks, including the prolonged time required for isolation and the limited number and size of samples which can be processed with an ultracentrifuge. Also, the high cost of an ultracentrifuge may be prohibitive in certain circumstances.
Another method for the simultaneous isolation of DNA, RNA and proteins from a single biological material sample is the subject of my earlier U.S. Pat. No. 5,346,994 (the '994 patent). That method is based on liquid-phase separation using phenol and guanidine thiocyanate. A biological sample is homogenized in the aqueous solution of phenol and guanidine thiocyanate and the homogenate thereafter is mixed with chloroform. Following centrifugation, the homogenate separates into an organic phase, an interphase and an aqueous phase. Proteins are sequestrated into the organic phase, DNA into the interphase and RNA into the aqueous phase. Next, each component is precipitated from the corresponding phase using ethanol and is then washed. The whole procedure can be completed in about 2-3 hours, and is especially useful for the isolation of high quality RNA from a variety of sources. One drawback to this method is the use of highly toxic phenol.
There are many known methods for the separate isolation of DNA, RNA and proteins from biological material; i.e., protocols for isolating a single one of these components from a sample. In typical DNA isolation methods, a biological sample is lysed in a lysing solution and then the DNA is isolated from the lysate according to any one of a variety of multi-step protocols, which may take from one hour to several days to complete. Frequently recommended DNA isolation methods involve the use of toxic phenol. See, Sambrook, J. et al., "Molecular Cloning", Vol. 2, pp. 9.14-9.23, Cold Spring Harbor Laboratory Press (1989) and Ausubel, Frederick M. et al., "Current Protocols in Molecular Biology", Vol. 1, pp. 2.2.1-2.4.5, John Wiley & Sons, Inc. (1994). Typically, a biological sample is lysed in a detergent solution and the protein component of the lysate is digested with proteinase for 12-18 hours. Next, the lysate is extracted with phenol to remove most of the cellular components, and the remaining aqueous phase is processed further to isolate DNA. In another method, described in Van Ness et al. U.S. Pat. No. 5,130,423, non-corrosive phenol derivatives are used for the isolation of nucleic acids. The resulting preparation is a mix of RNA and DNA.
DNA isolation methods utilizing non-corrosive chaotropic agents have also been developed. These methods, which are based on the use of guanidine salts, urea and sodium iodide, involve lysis of a biological sample in a chaotropic aqueous solution and subsequent precipitation of the crude DNA fraction with a lower alcohol. The final purification of the precipitated, crude DNA fraction can be achieved by any one of several methods, including column chromatography as described in "RapidPrep.TM. Genomic DNA Isolation Kits For Cells and Tissue: Versatility at Your Fingertips|", Analects, Vol 22, No. 4, Pharmacia Biotech, 1994, or exposure of the crude DNA to a polyanion-containing protein as described in Koller U.S. Pat. NO. 5,128,247.
Yet another method of DNA isolation, which is described in Botwell, D. D. L., "Rapid Isolation of Eukaryotic DNA", Anal. Biochem. 162, 463-465 (1987) involves lysing cells in 6M guanidine hydrochloride, precipitating DNA from the lysate at acid pH by adding 2.5 volumes of ethanol, and washing the DNA with ethanol. It is believed that the resulting DNA may be contaminated, however, with a low molecular weight material such as RNA and pigments. This conclusion is in agreement with the well known report showing that under similar conditions RNA can be precipitated from cell or tissue lysate. See Chirgwin, J. M. et al., "Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonuclease", Dept. of Biochemistry and Biophysics, Univ. of California, Vol. 18, No. 24, pp. 5294-5299 (1979).
Sodium iodide, another chaotropic agent, has been used in DNA isolation, but its use requires an additional purification step consisting of adsorption of the isolated DNA on glass beads. Although this method is relatively simple, it results in a low yield of isolated DNA.
In still another approach, the bulk of cytoplasmic proteins and RNA are removed by lysing cell samples in a detergent solution. The lysate is then fractionated into the nuclear and cytoplasmic fractions. And thereafter, the DNA is purified by dissolving the nuclear fraction in a chaotropic solution, precipitating and washing with ethanol. This method, described in Ciulla, T. A. et al., "A Simple Method for DNA Purification from Peripheral Blood", Anal. Biochem. 174, 485-488 (1988), can be completed in about 2 hours and is useful for isolating DNA from whole blood.
Known techniques for isolating RNA typically utilize either guanidine salts or phenol extraction, as described for example in Sambrook, J. et al., "Molecular Cloning", Vol. 1, pp. 7.3-7.24, Cold Spring Harbor Laboratory Press (1989) and Ausubel, F. M. et al., "Current Protocols in Molecular Biology", Vol. 1, pp. 4.0.3-4.4.7, John Wiley & Sons, Inc. (1994). Phenol-based techniques are multi-step procedures requiring several hours or days to complete. Similarly, the guanidine-based RNA isolation methods require at least several hours and take many steps to complete. In my earlier U.S. Pat. No. 4,843,155, phenol and guanidine procedures were uniquely combined, resulting in a simple method of total RNA isolation which can be completed in 3 hours. The method of the '155 patent was further improved upon in my '994 patent, which allows for completion of the RNA isolation in about 1 hour.
There are also known techniques for the simultaneous isolation of DNA and RNA, as referenced in my earlier '994 patent, the disclosures of which are incorporated herein by reference. All of these techniques utilize phenol extraction as a necessary step for isolating RNA and DNA free of protein contamination.
Heretofore, it has been the commonly accepted view that precipitation of nucleic acids from chaotropic solvents does not result in pure nucleic acid preparations. Contrary to this view, however, the present inventor has found that under certain conditions, as described in full detail herein, the use of chaotropic agents alone will result in isolation of assay ready, high quality DNA and RNA. This unexpected finding led to the development of a very simple, effective method and product for the simultaneous isolation of DNA, RNA and proteins from a single sample for subsequent use in molecular biology, biotechnology, clinical research and other applications.